Efeito de moléculas de quorum sensing exógenas e terpinen-4-ol sobre células planctônicas e biofilmes de Sporothrix spp. e desenvolvimento de plugin para análise de biofilmes fúngicos

Abstract

Sporotrichosis, a fungal infection caused by dimorphic fungi belonging to the genus Sporothrix, is the most prevalent subcutaneous mycosis in the world. In addition to the limited pharmacological arsenal for the treatment of this disease, recent studies have shown that Sporothrix spp. it is capable of forming biofilms, a structure classically associated with increased tolerance to antifungals. Thus, terpinen-4-ol and quorum sensing molecules (MQSs) previously described with antifungal activity become interesting study molecules against biofilms from Sporothrix spp. Thus, the objective of this study was to evaluate the effects of terpinen-4-ol and quorum sensing farnesol, tyrosol, 2-phenylethanol and tryptophan molecules, on planktonic cells and biofilm formation, sensitivity and morphology of Sporothrix spp., in addition to developing software for quantification of fungal biofilms using Sporothrix spp. as an analysis model. In this study, 15 strains of Sporothrix spp. in its filamentous and yeast forms. Antifungal activity in planktonic cells was evaluated by means of a broth microdilution assay following the protocols M27-A3 and M38-Ed3 of the Clinical & Laboratory Standards Institute. In addition, we evaluated the terpinie-4-ol effect on the cell ergosterol content and its interaction with the antifungal amphotericin B, itraconazole and terbinafine using the checkerboard technique. The effect of these molecules on the formation of biofilm and / or mature biofilm was evaluated by biomass quantification and metabolic activity using the technique of violet crystal staining and XTT reduction assay, in addition to confocal and scanning electron microscopy techniques for analysis of the biofilm morphology. The development of the plugin was performed in Java language to analyze the fluorescence intensity of the Syto9 and propidium iodide markers. Terpinen-4-ol showed minimal inhibitory concentrations (MIC) ranging from 4 to 32 µg/ml, decreasing the cell ergosterol content by up to 72%. In the pharmacological interaction assay, terpinen-4-ol showed synergism with itraconazole and terbinafine. The MQSs showed CIMs in the range of 0.01-1 µM (farnesol), 1-8 mM (2- phenylethanol and tyrosol) and > 16 mM (tryptophan). In the biofilm formation tests, only farnesol, tyrosol and 2-phenylethanol caused changes in the biofilm formation. The formation of filamentous biofilm was inhibited by farnesol (0.016 µM) and 2-phenylethanol (0.125 mM) and stimulated by tyrosol (1 mM), while the formation of biofilm in the yeast form was inhibited by 2-phenylethanol (0.125 mM) and tyrosol (0.125 mM). In the stage of evaluation of the sensitivity of mature biofilms, terpinen-4-ol presented a minimum inhibitory concentration in biofilm (CIMB) ranging from 64 to 1024 µg/ml, while the MQSs presented CIMB ranging from 8-32 µM (farnesol), 8- 32 mM (2-phenylethanol) and 64-128 mM (tyrosol). The performance tests of the QuantyFilm plugin showed that the analytical method generates results comparable to the XTT reduction test. Thus, it is concluded that terpinen-4-ol, farnesol, 2-phenylethanol and tyrosol have antifungal activity against planktonic and sessile cells of Sporothrix spp.; terpinen-4-ol reduces the ergosterol content in cells of Sporothrix spp. and presents synergism with classic antifungals; and that the exogenous MQSs modulate the biofilm formation of the filamentous forms and yeasts of Sporothrix spp.